Project I will continue to focus on the concept that epithelial regulation of the volume and/or composition of airway surface liquids (ASL) directly, and/or via mucociliary clearance (MCC), is crucial for airways defense. In this context, the grant proposes to study airway epithelial Na+ transport, focusing in part on the newly cloned epithelial Na+ channel (ENaC). The proposal widens its scope to include the study of a specific element in the airway epithelial lumenal domain, i.e., P2U receptor (P2U- R), that may be responsible for the coordination of ion transport activities, ciliary beat frequency, and secretory cell secretion to effect efficient mucociliary clearance (MCC). The major specific hypotheses to be tested are: 1) P2U-R is a central coordinator of airways MCC and defense against bacterial infection; and (2) regulation of airway epithelial Na+ transport rates is critical for the regulation of ASL volume/composition, MCC, and bacterial defense. The general strategy proposed to test each of these hypotheses involves manipulation of the level of gene expression of each of these elements in mice. Homologous recombination (knockout) techniques will be used to disrupt the genes for P2U-R and the three (alpha, beta, gamma) ENaC subunits to test for the contribution of each element to airway mucosal physiology/defense via a "loss of function" approach. In contrast, the role of each of these elements will be tested for their contribution to airway mucosal physiology/defense using overexpression transgenic approaches to test for consequences via a "gain of function" strategy. Specific interactions relevant to lung infectious phenotype between mice devoid of the P2U-R receptor [P2U-R(-/-) and mice overexpressing ENaC (alpha, beta, gamma) with mice devoid of CFTR [CFTR(-/-)] will be tested. The project investigators have developed techniques to measure the outcome of these genetic manipulations in mice at the whole animal levels, and in the lung at the "microscopic" (ion transport across superficial and gland epithelia, ciliary beat frequency, secretory cell function) and "macroscopic" scale (pulmonary function, MCC, and bacterial clearance). The goal of the Project is to test precisely the contribution of each of these elements to the physiology of the mucosa and ultimately to mucosal defense. These data should allow a better understanding of non-CF lung diseases that exhibit a phenotype of bacterial airways infection, for which etiologies are not determined, and elucidate novel pathways for therapeutic interventions to promote normalization of airways MCC function and bacterial clearance.